Theophylline (1,3-dimethylxanthine) is a commonly use drug in the treatment of bronchial asthma in adults and children. Assays for theophylline are important because patients show a variable response to standard dosage regimens and because there is a relatively small dosage range between sub-therapeutic and toxic doses.
Caffeine (1,3,7-trimethylxanthine) is a commonly encountered exogenous substance in human blood. It is structurally similar to theophylline except for the presence of the 7-methyl group. Anitibodies raised to theophylline are often unable to distinguish between caffeine and theophylline and hence may react with both. This phenomenon of cross-reactivity is undesirable in an assay for theophylline since it may result in values higher than actual. Therefore, in an immunoassay for theophylline, the removal of caffeine interference is necessary.
At present, the most commonly employed immunological assays for theophylline depend on antibody specificity to reduce caffeine interference. The need to produce specific antibodies is a disadvantage, however, since the necessary specific immunogens are often difficult or expensive to synthesize and since reproducible, specific antibodies are difficult to elicit from animals.
Another method for measuring theophylline and caffeine utilizes liquid chromatography. A variety of separation methods and column materials is known in the art. These suffer from several disadvantages for routine clinical assays since they require specialized, expensive equipment and have to employ organic solvents which are not compatible either with the biological samples or with assay systems. This incompatibility requires a separate extraction step to free the theophylline and caffeine from the serum sample.
The literature abounds with references to the adsorption of organic species by high surface area hydrophobic styrene-divinylbenzene copolymers and the wetting of their surfaces by organic solvents prior to use; see, for example, U.S. Pat. No. 3,531,463, issued Sept. 29, 1970 and Pranitis, et al., Journal of Forensic Sciences, Volume 19, 917 (1974).
The subsequent elution of the adsorbed species is also commonplace. Separation of the adsorbed species, however, usually has to be accomplished by subsequent chromatographic techniques, often involving step wise or gradient elution rather than isocratic elution and often requiring organic solvents.
An Application Note by E. I. du Pont de Nemours and Company for the PREP.TM. I Automated Sample Processor discloses a two-step method for measuring quantities of theophylline in human serum and plasma. The first step is an extraction of the lipophilic components, including theophylline and caffeine, utilizing a styrene-divinylbenzene copolymer resin. The second step is reverse phase high pressure liquid chromatography during which the separation of caffeine and the photometric measurement of theopylline occur. Both of these steps require organic solvents.
There is a need for a simple column technique for reducing caffeine interference in a theophylline immunoassay by an aqueous, isocratic elution of theophylline without the concomitant elution of caffeine.